Molecular determinants of the p<i>K</i><sub>a</sub> values of Asp and Glu residues in staphylococcal nuclease

Castañeda, Carlos; Fitch, Carolyn A.; Majumdar, Ananya; Khangulov, V.S.; Schlessman, J.L.; García-Moreno, Bertrand

doi:10.1002/prot.22470

Cited by 161 publications

(335 citation statements)

References 85 publications

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“…3C). One crystallographic water molecule occupies a small recess extending inwards between β-1 and α-1; an analogous water molecule was observed previously in the structure of the Δ+PHS protein (16). Other than this semiburied water molecule, there are no polar atoms within 4.5 Å of either carboxyl oxygen of the Glu side chain, leaving several potential hydrogen bonds unsatisfied.…”

Section: Resultssupporting

confidence: 54%

“…In these models the favorable Coulomb interaction between the charges in the buried pair is never strong enough to offset the unfavorable self-energy experienced by the two buried charges. In some microscopic models, Coulomb interactions can be stronger than dehydration effects, but calculations with these methods are still suspect as they cannot reproduce self-energies reliably (14, 15) and tend to overestimate the magnitude of Coulomb interactions (14,(16)(17)(18). The experiments that were performed test assumptions made in most continuum methods, including the idea that a pair of ionizable groups interacting at close range in a dehydrated environment can be treated classically as point charges without invoking some level of quantum mechanical treatment to account for polarizability and for the properties of a proton shared between acidic and basic moieties interacting over a short distance in a highly dehydrated environment (19,20).…”

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confidence: 99%

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Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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An artificial charge pair buried in the hydrophobic core of staphylococcal nuclease was engineered by making the V23E and L36K substitutions. Buried individually, Glu-23 and Lys-36 both titrate with pK a values near 7. When buried together their pK a values appear to be normal. The ionizable moieties of the buried Glu-Lys pair are 2.6 Å apart. The interaction between them at pH 7 is worth 5 kcal/mol. Despite this strong interaction, the buried Glu-Lys pair destabilizes the protein significantly because the apparent Coulomb interaction is sufficient to offset the dehydration of only one of the two buried charges. Save for minor reorganization of dipoles and water penetration consistent with the relatively high dielectric constant reported by the buried ion pair, there is no evidence that the presence of two charges in the hydrophobic interior of the protein induces any significant structural reorganization. The successful engineering of an artificial ion pair in a highly hydrophobic environment suggests that buried Glu-Lys pairs in dehydrated environments can be charged and that it is possible to engineer charge clusters that loosely resemble catalytic sites in a scaffold protein with high thermodynamic stability, without the need for specialized structural adaptations.electrostatics | internal charge | low-barrier hydrogen bond | dielectric constant | enzyme design P aired ionizable groups buried in hydrophobic environments inside proteins are an uncommon but essential structural motif necessary for H + transport (1), e − transfer (2), catalysis (3-5), and other important biochemical processes. Despite their importance, the properties of these buried ionizable pairs are poorly understood because they are difficult to study experimentally in the proteins where they play functional roles. When the pair consists of a basic and an acidic group it is usually assumed to be charged, but this is often just speculation without direct experimental support (6, 7). In principle, a Coulomb interaction between two ionizable groups of opposite polarity buried in a dehydrated environment inside a protein can be very strong, but this has not been established experimentally. Simulations suggest that buried ion pairs are always destabilizing (8); however, from an experimental perspective it is not known if the favorable Coulomb interaction would be sufficient to compensate for the unfavorable dehydration of the two buried charges or if the net effect of the pair on the thermodynamic stability of a protein would be stabilizing or not. These issues were examined in detail with an artificial ion pair buried in the hydrophobic interior of staphylococcal nuclease (SNase).The buried ion pair was engineered by introducing the V23E and L36K substitutions in a highly stable form of SNase. The pK a values of Glu-23 and Lys-36 introduced individually are anomalous (pK a of 7.1 for ref. 9; ref. 10), consistent with the groups existing in hydrophobic environments that are neither as polar nor as polarizable as water (11-13). Structural modeli...

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Section: Resultssupporting

confidence: 54%

mentioning

confidence: 99%

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Robinson

Castañeda

Schlessman

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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“…pK a measurements report native state electrostatic energetics and are a sensitive probe of the effect of mutations (35). The pK a values are identical in the two proteins, indicating that the mutation does not perturb native state electrostatic interactions involving the acidic side chains (Table S4) (21,24 The thermodynamic studies indicate that K12 is coupled to a multitude of hydrophobic sites.…”

Section: Resultsmentioning

confidence: 99%

Energetically significant networks of coupled interactions within an unfolded protein

Cho

Meng

Sato

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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Unfolded and partially unfolded proteins participate in a wide range of biological processes from pathological aggregation to the regulation of normal cellular activity. Unfolded states can be populated under strongly denaturing conditions, but the ensemble which is relevant for folding, stability, and aggregation is that populated under physiological conditions. Characterization of nonnative states is critical for the understanding of these processes, yet comparatively little is known about their energetics and their structural propensities under native conditions. The standard view is that energetically significant coupled interactions involving multiple residues are generally not present in the denatured state ensemble (DSE) or in intrinsically disordered proteins. Using the N-terminal domain of the ribosomal protein L9, a small α-β protein, as an experimental model system, we demonstrate that networks of energetically significant, coupled interactions can form in the DSE of globular proteins, and can involve residues that are distant in sequence and spatially well separated in the native structure. X-ray crystallography, NMR, dynamics studies, native state pK a measurements, and thermodynamic analysis of more than 25 mutants demonstrate that residues are energetically coupled in the DSE. Altering these interactions by mutation affects the stability of the domain. Mutations that alter the energetics of the DSE can impact the analysis of cooperativity and folding, and may play a role in determining the propensity to aggregate.protein folding | protein stability | ϕ-value analysis | double-mutant cycle T he properties of nonnative states of proteins have attracted considerable attention because they can play critical roles in cell signaling, translocation across membranes, temperaturesensitive phenotypes, and in a wide range of protein deposition diseases (1-14). The properties of unfolded states are also relevant to any discussion of intrinsically disordered proteins (IDPs) (1). The denatured state ensemble (DSE) can be populated under strongly denaturing conditions and studied at equilibrium; however, the physiologically relevant DSE is the state that is in equilibrium with the folded state under native conditions. This state is difficult to study directly and most studies of the DSE under native conditions have made use of indirect approaches. Nevertheless, experimental and theoretical work has shown that the DSE populated under physiological conditions is not a random coil, but can contain native and nonnative structure, stabilized by local and nonlocal interactions. However, very little is known about the energetics of these interactions, particularly ones involving residues distant in sequence, or about energetically coupled interactions involving multiple residues in the . These are a critical piece of the folding puzzle; they impact protein aggregation and folding, and have important implications for the properties of IDPs. Recent studies offer evidence for hydrophobic clusters, electrostatic interactio...

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“…A theoretical calculation has shown that Glu451 presents an unusually high pK a of 9.9 (Bello et al, 2012), which is probably the result of interactions with other carboxylic groups at the TNC (Harris & Turner, 2002;Castañ eda et al, 2009). Accordingly, the perturbed pK a value of Glu451 seems to be governed by its interaction with the Asp452 residue (pK a = 4.40, as calculated using the PROPKA server; Olsson et al, 2011), which is at a hydrogen-bonding distance of 3.14 Å from Glu451a O "2 (Fig.…”

Section: Methionine-rich B-hairpin Motifmentioning

confidence: 99%

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O₂-reduction states

Serrano‐Posada

Centeno-Leija

Rojas-Trejo

et al. 2015

Acta Crystallogr D Biol Crystallogr

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PDB references: Tth-MCO, 2xu9; apo Tth-MCO, 2xuw; Hg-Tth-MCO, 2xvb; Hg-Tth-MCO-2h, 4ai7; Tth-MCO-C1, 2yae; Tth-MCO-C2, 2yaf; Tth-MCO-C3, 2yah; Tth-MCO-C4, 2yam; Tth-MCO-C5, 2yao; Tth-MCO-C6, 2yap; Tth-MCO-C7, 2yaq; Tth-MCO-C8, 2yar During X-ray data collection from a multicopper oxidase (MCO) crystal, electrons and protons are mainly released into the system by the radiolysis of water molecules, leading to the X-ray-induced reduction of O 2 to 2H 2 O at the trinuclear copper cluster (TNC) of the enzyme. In this work, 12 crystallographic structures of Thermus thermophilus HB27 multicopper oxidase (Tth-MCO) in holo, apo and Hg-bound forms and with different X-ray absorbed doses have been determined. In holo Tth-MCO structures with four Cu atoms, the protondonor residue Glu451 involved in O 2 reduction was found in a double conformation: Glu451a ($7 Å from the TNC) and Glu451b ($4.5 Å from the TNC). A positive peak of electron density above 3.5 in an F o À F c map for Glu451a O "2 indicates the presence of a carboxyl functional group at the side chain, while its significant absence in Glu451b strongly suggests a carboxylate functional group. In contrast, for apo Tth-MCO and in Hg-bound structures neither the positive peak nor double conformations were observed. Together, these observations provide the first structural evidence for a proton-relay mechanism in the MCO family and also support previous studies indicating that Asp106 does not provide protons for this mechanism. In addition, eight composite structures (Tth-MCO-C1-8) with different X-ray-absorbed doses allowed the observation of different O 2 -reduction states, and a total depletion of T2Cu at doses higher than 0.2 MGy showed the high susceptibility of this Cu atom to radiation damage, highlighting the importance of taking radiation effects into account in biochemical interpretations of an MCO structure.

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Molecular determinants of the pK_a values of Asp and Glu residues in staphylococcal nuclease

Cited by 161 publications

References 85 publications

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Energetically significant networks of coupled interactions within an unfolded protein

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O₂-reduction states

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Molecular determinants of the pKa values of Asp and Glu residues in staphylococcal nuclease

Cited by 161 publications

References 85 publications

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Structural and thermodynamic consequences of burial of an artificial ion pair in the hydrophobic interior of a protein

Energetically significant networks of coupled interactions within an unfolded protein

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O2-reduction states

Contact Info

Product

Resources

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Molecular determinants of the pK_a values of Asp and Glu residues in staphylococcal nuclease

X-ray-induced catalytic active-site reduction of a multicopper oxidase: structural insights into the proton-relay mechanism and O₂-reduction states